This invention relates to the fields of molecular biology and bone physiology. More specifically, the invention provides novel compositions and methods for enhancing osseous growth, repair and regeneration.
Several publications are referenced in this application by numerals in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Bone growth and development is a complex process involving the participation of many genes and proteins. The normal development of hair, teeth and bone involves extensive epithelial-mesenchyme interactions. For example, the tooth develops through interactions between oral ectoderm of epithelial origin which eventually produces enamel, while mesenchyme cells from the neural crest produce dentin and supportive structures (10).
Studies in vertebrate organisms have implicated the involvement of a number of genes in craniofacial development, including transcription factor and growth factor genes (10). Several homeobox genes are also known to be expressed in the developing teeth. These genes are postulated to function together with growth factors, such as bone morphogenic proteins and members of the transforming growth factor-beta genes, in the complex developmental pathways involved in craniofacial growth and development.
The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate variants thereof, that participate in the control of mammalian bone growth, development and regeneration.
Provided herein are novel compositions and methods for enhancing osseous growth, repair and regeneration. According to one aspect of the invention, an isolated nucleic acid molecule is provided which includes a complete coding region for a homeobox domain-containing DLX3 protein of a predicted size between about 25 and 35 kilodaltons.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human DLX3 protein. In a particularly preferred embodiment, the human DLX3 protein has an amino acid sequence the same as Sequence I.D. No. 2. An exemplary DLX3 encoding nucleic acid molecule of the invention comprises Sequence I.D. No. 1.
According to another aspect of the invention, another isolated nucleic acid molecule is provided which includes a complete coding region for a DLX3xcex94 protein also containing a homeobox domain of a size between about 20 and 35 kilodaltons. DLX3xcex94 appears to be a mutated form of DLX3 associated with Tricho-Dento-Osseous (TDO) syndrome. The rare genetic polymorphism present in DLX3xcex94 is correlated with the expression of a DLX3xcex94 protein, which is associated with increased bone thickness and density.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human DLX3xcex94 protein. In a particularly preferred embodiment, the human DLX3xcex94 protein has an amino acid sequence the same as Sequence I.D. No. 4. An exemplary dlx3xcex94 nucleic acid molecule of the invention comprises Sequence I.D. No. 3.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) Sequence I.D. No. 1; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 1; (3) a sequence encoding preselected portions of Sequence I.D. No. 1, (4) a sequence encoding part or all of a polypeptide having amino acid Sequence I.D. No. 2; (5) Sequence I.D. No. 3; (6) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 3; (7) a sequence encoding preselected portions of Sequence I.D. No. 3, and (8) a sequence encoding part or all of a polypeptide having amino acid Sequence I.D. No. 4. Such partial sequences are useful as probes to identify and isolate homologues of the DLX3 genes of the invention. Accordingly, isolated nucleic acid sequences encoding natural allelic variants of the nucleic acids of Sequence I.D. Nos., 1, or 3 are also contemplated to be within the scope of the present invention. The term xe2x80x9cnatural allelic variantsxe2x80x9d will be defined hereinbelow.
In another aspect of the invention, a nucleic acid molecule comprising the upstream 5xe2x80x2 untranslated region of the DLX3 gene is provided herein as Sequence I.D. NO: 5. This sequence provides regulatory elements that control the expression of the DLX3 proteins of the invention. Accordingly, the sequence may be genetically manipulated using recombinant DNA tecniques known to those of skill in the art to enhance or inhibit the expression of DLX3 proteins.
According to another aspect of the invention, antibodies immunologically specific for the proteins described hereinabove are provided.
In addition, this invention presents methods for genetic screening and diagnostic evaluation of patients at risk for TDO. Additionally, the nucleic acids of the invention may be used for prenatal genetic screening. The hybridization specificity of the nucleic acids of the invention may be also be used for differential evaluation of patients presenting with phenotypic characteristics commmon to TDO, such as amelogenesis imperfecta. For example, nucleic acid molecules of the invention can be used as diagnostic hybridization probes or as primers for diagnostic PCR analysis for the identification of DLX3 mutations. Additionally, human DLX3 genomic sequences are provided which can be used to selectively amplify human DLX3 exons for analysis.
Further, methods and compositions are provided for enhancing growth, development and repair of osseous structures. Addition of DLX3xcex94 proteins and DLX3xcex94-encoding nucleic acids should serve to enhance bone thickness and increase bone density at the sites of application. Exogenously added DLX3xcex94 proteins and DLX3xcex94-encoding nucleic acids should have utility in the treatment of bone/osseous defects secondary to trauma, such as broken bones. Finally, the DLX3xcex94 proteins and nucleic acids of the invention should also have utility in the treatment of defects secondary to certain pathologies, such as periodontal disease defects or congenital/acquired defects such as osteoporosis.